Fluorescence instrumentation has been used for many years to identify unknown materials. Generally, the principle involved is that a material excited with light of a particular wavelength will emit light energy in the form of an emission spectrum whose amplitude profile, over the range of wavelengths emitted, constitutes a xe2x80x9cfingerprintxe2x80x9d which can give the identity and nature of the unknown material.
In the most demanding applications, a sample is excited with light of a single wavelength and the fluorescence emission spectrum is recorded. The wavelength of the excitation source is then advanced incrementally along the range of excitation wavelengths of interest, and the process repeated to record the fluorescence emission spectrum at the incremented wavelength. The process is continued until the entire range of excitation wavelengths of interest has been covered by the instrument. The result is a highly accurate, so-called three-dimensional fluorescence emission spectrum, showing excitation wavelengths, corresponding emission wavelengths and their amplitudes. Such instruments are of particular interest in scientific research where subtle variations in the characteristics of the spectrum may contain useful information to understand the effects of relatively subtle changes in the system. Typically, instruments of this sort have resolutions on the order of between 0.1 to 0.5 nm.
However, many applications have far less demanding requirements. For example, if one is merely interested in identifying the identity of a particular sample of material, far less resolution will suffice. Accordingly, a class of instruments having resolutions on the order of five to ten nanometers have seen widespread application in industry. Typical applications include the identification of samples of such material as blood, oil, pollutants and the like. Such instruments differ from other fluorescence instruments in that they are designed to perform measurements much more quickly, by measuring the fluorescence of a material over a range of wavelengths simultaneously.
Such a prior art system is illustrated in FIG. 1. Measurement of the fluorescence spectrum is achieved by having a system which comprises an excitation spectrograph 1 which is used to excite a sample 2, typically contained in. an elongated cuvette 3. The elongated cuvette 3 is excited by an elongated image of a spectrum extending from a low wavelength to a high wavelength.
This results in fluorescence emission by sample 2 in cuvette 3. The emission is received and collimated by a collimating concave mirror 4, which reflects the fluorescence emission to focusing concave mirror 5, which, in turn, focuses the emitted fluorescence light at a slit 6, through which the light which comprises the fluorescence emission passes to fall on the planar mirror 7. Planar mirror 7 reflects the light toward a spectrograph 8 formed by a concave aberration-corrected diffraction grating. Spectrograph 8 disperses a spectrum on a CCD detector 9 which in a single row of pixels can produce the complete emission spectrum of the excited material.
In a typical instrument of this type, a xenon source is dispersed as a spectrum placed over a cuvette along a vertical axis. Thus, the full spectrum will excite any homogeneous sample placed in the sample compartment of the cuvette. The resulting fluorescence emission is dispersed orthogonally over the active area of a rectangular CCD, or charge-coupled device, which is, essentially, a two-dimensional array of light detectors. The horizontal axis of the CCD records the emission spectra at different excitation wavelengths along the vertical axis, and gives the intensity for each wavelength. Thus, this instrument will produce, for each wavelength in the range of excitation wavelengths, the spectrum of emitted wavelengths. For example, if the system has a resolution of 5 nm, and covers a range of 100 nm, one could view the output as twenty different spectra.
The abiliby to complete a reading of the emission spectrum simultaneously opens up many possibilities for enhanced performance functions. For example, a cuvette may be fed by a high pressure liquid chromatography column, allowing the facile real-time generation of fluorescence emission spectra of the various materials in a sample being analyzed by the chromatography column.
While this system has many advantages over the prior art systems which measured a fluorescence spectrum one wavelength at a time, it still had a number of deficiencies. First, the volume required for the system is relatively large and precludes use of the system in a compact system. Moreover, the system comprises numerous expensive parts, and costs may be prohibitive for many applications. In addition, assembly of the system is unduly expensive requiring careful alignment of parts to ensure proper operation of the system. Similarly, the system is not as rugged as other systems, and is liable to become misaligned during use on account of shock and vibration. Finally, the system is limited to producing a fluorescence spectrum.
The invention, as claimed, is intended to provide a remedy. It solves the problems of large size, lack of ruggedness and cost by providing a simple instrument that can be implemented in a compact design. In accordance with the present invention, an excitation light source provides optical radiation over a range of wavelengths or spectra for illuminating a sample. The inventive instrument performs fluoresence analysis of samples, and comprises a light source emitting light into an illumination light path, and a first spectral filter in the illumination light path for transmitting light within a selected wavelength range. This defines a sample illumination light path. A second spectral filter is spaced from the first spectral filter forming a sample receiving space therebetween.
The illumination light path passes through the first spectral filter. The sample receiver and the second spectral filter lie in the light path, and the second spectral filter is displaced angularly relative to the first spectral filter. A sensing element in the resultant light path measures absorption spectra and fluorescence light. The first spectral filter and the second spectral filter have a characteristic which varies along an axis thereof. In accordance with the preferred embodiment of the invention, the variable characteristic is a variable bandpass wavelength in various filter regions of the spectral filter.
Also in accordance with the preferred embodiment, the second spectral filter is angularly displaced at a substantially othogonal angle.
The above described embodiment of the invention has the advantage of providing along a diagonal region of the CCD the absorption spectrum of the material sample under analysis.
In accordance with an alternative embodiment of the invention, a third spectral filter in the resultant light path is oriented in a direction, and position in a position which are substantially the same as the direction and position of the first spectral filter. This third filter serves the function of a blocking filter thereby preventing excitation light energy that has passed through a sample receiver from passing to the sensing element or CCD array.